Tumor metastasis is the leading cause of cancer deaths. Metastatic cancer cells use invadopodia, specialized filament-like membrane protrusions, to degrade and invade through surrounding extracellular matrix (ECM). However, invadopodia-mediated invasion remains understudied, and little is known about the molecular mechanisms that govern invadopodia formation and function. My long-term goal is to elucidate mechanisms of invadopodia-mediated invasion. This application has the dual objective of: (1) providing training required for my transition from postdoctoral fellow to independent successful scientist in the field of cancer biology/metastasis, and (2) determining the cellular mechanisms of invadopodia induction upon tumor cell adhesion to ECM, which is the next logical step in pursuit of my research goal. The central hypothesis of this proposal is that invadopodia assembly is initiated by signaling events at unique cell adhesions of invading cancer cells that we call preinvadopodial adhesions. This hypothesis stems from strong preliminary data, and was formulated on the basis of the unique live-cell model system that I developed in our laboratory for simultaneous visualization and analysis of invadopodia formation and function in ECM degradation [unreadable] (Artym et al., Cancer Res. 66:3034-43, 2006). Using three-channel live-cell imaging, I recently discovered that first, structural cores of invadopodia rich in actin and cortactin are formed, and then, MT1-MMP, a metalloprotease required for invadopodia function, is accumulated at the invadopodia triggering ECM degradation. I have now observed invadopodia initiation and formation at specific adhesion sites, preinvadopodial adhesions. [unreadable] [unreadable] To test the central hypothesis and to accomplish the objective of this application, three specific aims are proposed: 1) Determine the mechanism of invadopodia regulation by p1 and (33 integrins; 2) Determine the function of vinculin in invadopodia formation; 3) Determine the role of ECM in regulation of invadopodia induction from preinvadopodial adhesions in physiologically relevant 3D in vitro system. Confocal and TIRF microscopy techniques in combination with siRNA technology, protein biochemistry, and electron microscopy will be used. The proposed work is innovative because it takes advantage of the real-time live-cell model developed by me in our laboratory that allows characterizing protein dynamics at invadopodia while simultaneously monitoring invadopodia assembly and function. The proposed research is significant because it will expand our knowledge of malignant transformation and provide potential targets for control of tumor cell invasion and metastasis. Relevance to Public Health: understanding the mechanisms governing the formation and function of invadopodia should provide insights into tumor cell biology that could lead to potential therapeutic approaches to cancer metastasis. [unreadable] [unreadable] [unreadable] [unreadable]